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Short answer
FITC is fluorescein isothiocyanate, a green fluorescent dye used to label proteins such as antibodies. It is excited by the 488 nm laser line and emits near 519 nm. Its main limits are that its fluorescence falls below pH 7 and that it photobleaches faster than newer dyes such as Alexa Fluor 488.
Definition and scope
FITC is a reactive derivative of fluorescein. The isothiocyanate group lets the dye bind covalently to primary amines, so it can be attached to proteins, most often antibodies used in flow cytometry and immunofluorescence. In practice, “FITC” is also used as the name of a detection channel: the green channel read with a 488 nm laser and a filter around 530 nm. A different dye measured in that channel is not FITC, even though instruments and panels may still label the channel that way.
The Molecular Probes Handbook (Thermo Fisher) lists FITC with an absorption maximum of 494 nm and an emission maximum of 519 nm, measured at pH 9, and notes that the excitation maximum closely matches the 488 nm spectral line. Other vendor pages round the figures slightly differently (for example 494/518 nm on the Pierce protein methods page, and 490/525 nm on Cell Signaling Technology’s page), so published values vary by a few nanometres depending on the source and conditions. This site’s fluorophores guide places FITC among small organic dyes.
How FITC labels proteins and is detected
Labeling chemistry
According to Thermo Fisher’s Pierce protein methods page, FITC is an isothiocyanate that reacts with primary amines, such as lysine side chains, at pH 7.0 to 9.0,. The same page says NHS-ester chemistry is now the preferred method for amine labeling because it is more specific for primary amines and gives a more stable linkage.
The Molecular Probes Handbook describes a study comparing several reactive fluorescein derivatives. The succinimidyl ester of carboxyfluorescein showed the best performance, followed by fluorescein dichlorotriazine (DTAF); FITC was both the slowest to react and yielded the least stable conjugates, although the degree of labeling was most easily controlled with FITC. Succinimidyl esters of carboxyfluorescein yield carboxamides that are more resistant to hydrolysis.
Detection
Cell Signaling Technology describes FITC conjugates as read with the 488 nm laser line and a common 530/30 filter set, with excitation at 490 nm, emission at 525 nm and a quantum yield of 0.50. That is a vendor description and the rating scales for brightness differ between vendors, so compare dyes using the same source rather than mixing scales.
FITC compared with other dyes in the 488 nm channel
Several dyes can be read in the same laser and filter combination. The table summarizes what each source states:
| Dye | What the source says | Source |
|---|---|---|
| FITC | Excitation maximum 494 nm closely matches the 488 nm line; relatively high photobleaching rate limits sensitivity; fluorescence significantly reduced below pH 7 (fluorescein pKa 6.4) | Molecular Probes Handbook |
| Alexa Fluor 488, BODIPY FL, Oregon Green, Rhodamine Green | Described as much more photostable than fluorescein | Molecular Probes Handbook |
| Oregon Green | pKa 4.7 for the dye, compared with 6.4 for fluorescein | Molecular Probes Handbook |
| Alexa Fluor 488, KIRAVIA Blue 520, SureLight 488 | Listed as related or alternative conjugates for FITC | Cell Signaling Technology |
Because dyes in this channel overlap spectrally, a panel that contains two of them needs careful controls. See the guide on flow cytometry compensation and single-stain controls, and the overview of flow cytometry dyes.
Where it matters in the lab: a workflow checklist
The points below follow FITC from reagent to image, with the source for each listed at the end of the page.
- Chemistry. FITC reacts with primary amines at pH 7.0 to 9.0; NHS-ester reagents are the preferred route for amine labeling, and FITC was the slowest to react and gave the least stable conjugates among the reactive fluorescein derivatives in the Molecular Probes comparison.
- Instrument. Excitation by the 488 nm laser line; a 530/30 filter set is the common detection configuration.
- Assay conditions. Fluorescein fluorescence is significantly reduced below pH 7 (pKa 6.4), so acidic buffers or acidic cell compartments lower the signal.
- Imaging. Photobleaching is the irreversible destruction of the dye by prolonged or high-intensity excitation. Thermo Fisher lists minimizing excitation intensity and exposure time, and using antifade reagents for fixed cells, as mitigations. See photobleaching and phototoxicity in live-cell imaging and immunofluorescence controls and troubleshooting.
Common misunderstandings
“FITC” and “fluorescein” are not the same thing
Fluorescein is the fluorophore. FITC is the isothiocyanate derivative of it that carries the amine-reactive group. NHS-fluorescein is a different amine-reactive derivative of the same fluorophore; Thermo Fisher gives both nearly the same spectra (494 nm excitation, 518 nm emission).
“FITC channel” does not mean FITC dye
The channel is defined by the laser and filters. Any green dye excited at 488 nm can be read there, and the dyes listed above differ in photostability and pH sensitivity.
Spectra are not one fixed pair of numbers
The Molecular Probes table (494/519 nm) is measured at pH 9. Other pages quote 490/525 or 494/518 nm. The practical point is the same: the 488 nm laser excites it efficiently.
FAQ
What does FITC stand for?
Fluorescein isothiocyanate: the amine-reactive derivative of the green fluorophore fluorescein.
What laser excites FITC?
The 488 nm line, such as an argon-ion laser. Thermo Fisher notes the 494 nm excitation maximum closely matches that line.
Why does FITC signal fade or drop?
Two documented causes: photobleaching, which is the irreversible loss of fluorescence under intense or prolonged excitation, and reduced fluorescence below pH 7.
What are alternatives to FITC in the same channel?
Thermo Fisher describes Alexa Fluor 488, BODIPY FL, Oregon Green and Rhodamine Green as much more photostable than fluorescein, and Cell Signaling Technology lists Alexa Fluor 488, KIRAVIA Blue 520 and SureLight 488 as related conjugates. For background on labeling in general, see immunostaining.
Sources
- Thermo Fisher Scientific, Molecular Probes Handbook, “Fluorescein, Oregon Green and Rhodamine Green Dyes” (Section 1.5): https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/fluorophores-and-their-amine-reactive-derivatives/fluorescein-oregon-green-and-rhodamine-green-dyes.html. Checked 2026-10-29.
- Thermo Fisher Scientific, Pierce protein methods, “Fluorescent probes”: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/fluorescent-probes.html. Checked 2026-10-29.
- Cell Signaling Technology, “FITC-conjugated antibodies” (vendor description): https://www.cellsignal.com/learn-and-support/antibody-conjugates/fitc-conjugated-antibodies. Checked 2026-10-29.
